Generation of plants with improved drought tolerance

ABSTRACT

The present invention is directed to plants that display a drought tolerance phenotype due to altered expression of a DR02 nucleic acid. The invention is further directed to methods of generating plants with a drought tolerance phenotype.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation of U.S. application Ser. No. 10/509,691, filedMay 8, 2005, now U.S. Pat. No. 7,432,416, issued on Oct. 7, 2008, whichis the National Stage of PCT/US2003/09479, filed on Mar. 27, 2003, whichclaims benefit of U.S. Application No. 60/368,650, filed Mar. 27, 2002.The entire disclosures of each of these applications are herebyexpressly incorporated by reference.

BACKGROUND OF THE INVENTION

Crop production is affected by numerous abiotic environmental factors,with soil salinity and drought having the most detrimental effects.Approximately 70% of the genetic yield potential in major crops is lostdue to abiotic stresses, and most major agricultural crops aresusceptible to drought stress. Attempts to improve yield under stressconditions by plant breeding have been largely unsuccessful, primarilydue to the multigenic origin of the adaptive responses (Barkla et al.1999, Adv Exp Med Biol 464:77-89).

Considerable effort has focused on the identification of genetic factorsthat contribute to stress tolerance and on the genetic engineering ofcrop plants with increased stress tolerance. A number of genes have beenidentified whose expression or misexpression is associated with droughttolerance, via a variety of different mechanisms. For instance,transformed tobacco that express maize NADP-malic enzyme displayincreased water conservation and gained more mass per water consumedthan wild-type plants (Laporte et al. 2002, J Exp Bot 53:699-705).Significant research effort has focused on the plant hormone abscisicacid (ABA), which is involved in adaptation to various environmentalstresses. Transgenic tobacco and transgenic Arabidopsis that overexpressthe enzyme 9-cis-epoxycarotenoid dioxygenase (NCED), which is key to ABAbiosynthesis, display improved drought tolerance (Qin et al. 2002, PlantPhysiol 128:544-51; Iuchi et al. 2001, Plant J 27:325-33). Droughttolerance is often linked to salt tolerance, since both are associatedwith regulation of osmotic potential and turgor. Accordingly, transgenicplants that overexpress a vacuolar H+ pump (H+ pyrophosphatase), whichgenerates a proton gradient across the vacuolar membrane, displayimproved drought- and salt-stress, due to increased solute accumulationand water retention (Gaxiola et al. 2001, Proc Natl Acad Sci USA98:11444-9). Trehalose also contributes to osmoprotection againstenvironmental stress. Potato plants the mis-expresstrehalose-6-phosphate synthase, a key enzyme for trehalose biosynthesis,show increased drought resistance (Yeo et al. 2000, Mol Cells 10:263-8).

Arabidopsis has served as a model system for the identification of genesthat contribute to drought tolerance. For instance, researchers haveidentified numerous genes that are induced in response to waterdeprivation (e.g., Taji et al. 1999, Plant Cell Physiol 40:119-23;Ascenzi et al., 1997, Plant Mol Biol 34:629-41; Gosti et al. 1995, MolGen Genet 246:10-18; Koizumi et al. 1993 Gene 129:175-82) and cis-actingDNA sequences called ABA responsive elements (ABREs) that control ABA orstress responsive gene expression (Giraudat et al. 1994, Plant Mol.Biol. 26:1557).

Several drought tolerant mutants of Arabidopsis have been identified.These include the recessive mutants abh1 (Hugouvieux et al. 2001, Cell106: 477), era1-2 (Pei et al. 1998, Science 282: 286) and abi1-1Ri(Gosti et al. 1999, Plant Cell 11:1897-1909). The mutants era1-2 andabh1 were identified by screening for seedlings hypersensitive to ABA,while the mutant abi1-1 Ri was isolated as an intragenic suppressor ofthe ABA insensitive mutant abi1-1. Dominant drought tolerant mutantswere identified by overexpressing ABF3, ABF4 (Kang et al. 2002, PlantCell 14:343-357) or DREB1A (Kasuga, 1999 Nature Biotech 17: 287). ABF3and ARF4 encode basic-region leucine zipper (bZIP) DNA binding proteinsthat bind specifically ABREs. DREB1A encodes a protein with an EREBP/AP2DNA binding domain that binds to the dehydration-responsive element(DRE) essential for dehydration responsive gene expression (Liu et al.1998, Plant Cell 10:1391). A dominant drought tolerant phenotype intobacco was obtained by overexpressing the soybean BiP gene (Alvim etal. 2001, Plant Physiol 126, 1042).

Activation tagging in plants refers to a method of generating randommutations by insertion of a heterologous nucleic acid constructcomprising regulatory sequences (e.g., an enhancer) into a plant genome.The regulatory sequences can act to enhance transcription of one or morenative plant genes; accordingly, activation tagging is a fruitful methodfor generating gain-of-function, generally dominant mutants (see, e.g.,Hayashi H et al., Science (1992) 258:1350-1353; Weigel D, et al., PlantPhysiology (2000) 122: 1003-1013). The inserted construct provides amolecular tag for rapid identification of the native plant whosemis-expression causes the mutant phenotype. Activation tagging may alsocause loss-of-function phenotypes. The insertion may result indisruption of a native plant gene, in which case the phenotype isgenerally recessive.

Activation tagging has been used in various species, including tobaccoand Arabidopsis, to identify many different kinds of mutant phenotypesand the genes associated with these phenotypes (Wilson K et al., PlantCell (1996) 8: 659-671, Schaffer R, et al., Cell (1998) 93: 1219-1229,Fridborg I et al., Plant Cell 11: 1019-1032, 1999; Kardailsky I et al.,Science (1999) 286: 1962-1965; Christensen S et al., 9th InternationalConference on Arabidopsis Research. Univ. of Wisconsin-Madison, Jun.24-28, 1998. Abstract 165).

We used activation tagging techniques to identify the associationbetween the Arabidopsis DNA binding protein OBF1 and drought tolerance.OBF1 was first identified in a screen designed to characterize factorsassociated with proteins that bind to octopine synthase (ocs) promoterelements (Zhang et al. 1995, Plant Cell 7:2241-2252). The ocs elementsare 20 bp DNA motifs that were originally found in the Agrobaeterium ocspromoter and were subsequently identified in the CaMV 35S promoter andin promoters of a class of plant stress-inducible genes such asglutathione S-transferase genes, which may be involved in responses tooxidative stress. Auxins and/or salicylic acid can induce genes with ocspromoter elements, possibly through changes in the redox potential. TheArabidopsis OBF4/OBF5 proteins are basic-region leucine zipper (bZIP)transcription factors that have been shown to interact with ocselements. OBP1 was identified by its ability to bind OBF4 and OBF5, evenin the absence of the target ocs elements.

The OPB1 protein sequence predicted by Zhang et al. is presented inGenbank entry GI 1212759 and is 13 amino acids shorter at the N-terminalend than the protein predicted by the Arabidopsis sequencing project(i.e., encoded by the same genomic sequence) and presented in G17630027.

OBP1 contains a highly conserved Dof domain, a zinc finger DNA-bindingdomain found in proteins from a number of plant species (Yanagisawa S,1996, Trends Plant Sci. 1:213-214). OBP1 is constitutively expressedboth in aerial portions of the plant and in roots; it is induced byauxin, salicylic acid, and cycloheximide (Kang and Singh, Plant J 21:329-339, 2000).

SUMMARY OF THE INVENTION

The present invention provides a method of producing an improved droughttolerance phenotype in a plant. The method comprises introducing intoplant progenitor cells a vector comprising a nucleotide sequence thatencodes or is complementary to a sequence encoding a DRO2 polypeptideand growing a transgenic plant that expresses the nucleotide sequence.In one embodiment, the DRO2 polypeptide has at least 50% sequenceidentity to the amino acid sequence presented in SEQ ID NO:2 andcomprises a DNA binding domain. In other embodiments, the DRO2polypeptide has at least 80% or 90% sequence identity to or has theamino acid sequence presented in SEQ ID NO:2.

The invention further provides plants and plant parts obtained by themethods described herein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise indicated, all technical and scientific terms usedherein have the same meaning as they would to one skilled in the art ofthe present invention. Practitioners are particularly directed toSambrook et al. Molecular Cloning: A Laboratory Manual (Second Edition),Cold Spring Harbor Press, Plainview, N.Y., 1989, and Ausubel F M et al.Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., 1993, for definitions and terms of the art. It is to be understoodthat this invention is not limited to the particular methodology,protocols, and reagents described, as these may vary.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

A “heterologous” nucleic acid construct or sequence has a portion of thesequence that is not native to the plant cell in which it is expressed.Heterologous, with respect to a control sequence refers to a controlsequence (i.e. promoter or enhancer) that does not function in nature toregulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell, by infection, transfection,microinjection, electroporation, or the like. A “heterologous” nucleicacid construct may contain a control sequence/DNA coding sequencecombination that is the same as, or different from a controlsequence/DNA coding sequence combination found in the native plant.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, which may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons) and non-transcribed regulatory sequence.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

As used herein, the term “gene expression” refers to the process bywhich a polypeptide is produced based on the nucleic acid sequence of agene. The process includes both transcription and translation;accordingly, “expression” may refer to either a polynucleotide orpolypeptide sequence, or both. Sometimes, expression of a polynucleotidesequence will not lead to protein translation. “Over-expression” refersto increased expression of a polynucleotide and/or polypeptide sequencerelative to its expression in a wild-type (or other reference [e.g.,non-transgenic]) plant and may relate to a naturally-occurring ornon-naturally occurring sequence. “Ectopic expression” refers toexpression at a time, place, and/or increased level that does notnaturally occur in the nonaltered or wild-type plant. “Under-expression”refers to decreased expression of a polynucleotide and/or polypeptidesequence, generally of an endogenous gene, relative to its expression ina wild-type plant. The terms “mis-expression” and “altered expression”encompass over-expression, under-expression, and ectopic expression.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

As used herein, a “plant cell” refers to any cell derived from a plant,including cells from undifferentiated tissue (e.g., callus) as well asplant seeds, pollen, progagules and embryos.

As used herein, the terms “native” and “wild-type” relative to a givenplant trait or phenotype refers to the form in which that trait orphenotype is found in the same variety of plant in nature.

As used herein, the term “modified” regarding a plant trait, refers to achange in the phenotype of a transgenic plant relative to the similarnon-transgenic plant. An “interesting phenotype (trait)” with referenceto a transgenic plant refers to an observable or measurable phenotypedemonstrated by a T1 and/or subsequent generation plant, which is notdisplayed by the corresponding non-transgenic (i.e., a genotypicallysimilar plant that has been raised or assayed under similar conditions).An interesting phenotype may represent an improvement in the plant ormay provide a means to produce improvements in other plants. An“improvement” is a feature that may enhance the utility of a plantspecies or variety by providing the plant with a unique and/or novelquality.

An “altered drought tolerance phenotype” refers to detectable change inthe ability of a genetically modified plant to withstand low-waterconditions compared to the similar, but non-modified plant. In general,improved (increased) drought tolerance phenotypes (i.e., ability to aplant to survive in low-water conditions that would normally bedeleterious to a plant) are of interest.

As used herein, a “mutant” polynucleotide sequence or gene differs fromthe corresponding wild type polynucleotide sequence or gene either interms of sequence or expression, where the difference contributes to amodified plant phenotype or trait. Relative to a plant or plant line,the term “mutant” refers to a plant or plant line which has a modifiedplant phenotype or trait, where the modified phenotype or trait isassociated with the modified expression of a wild type polynucleotidesequence or gene.

As used herein, the term “T1” refers to the generation of plants fromthe seed of T0 plants. The T1 generation is the first set of transformedplants that can be selected by application of a selection agent, e.g.,an antibiotic or herbicide, for which the transgenic plant contains thecorresponding resistance gene. The term “T2” refers to the generation ofplants by self-fertilization of the flowers of T1 plants, previouslyselected as being transgenic.

As used herein, the term “plant part” includes any plant organ ortissue, including, without limitation, seeds, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores. Plant cells can be obtained fromany plant organ or tissue and cultures prepared therefrom. The class ofplants which can be used in the methods of the present invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledenous anddicotyledenous plants.

As used herein, “transgenic plant” includes reference to a plant thatcomprises within its genome a heterologous polynucleotide. Theheterologous polynucleotide can be either stably integrated into thegenome, or can be extra-chromosomal. Preferably, the polynucleotide ofthe present invention is stably integrated into the genome such that thepolynucleotide is passed on to successive generations. A plant cell,tissue, organ, or plant into which the heterologous polynucleotides havebeen introduced is considered “transformed”, “transfected”, or“transgenic”. Direct and indirect progeny of transformed plants or plantcells that also contain the heterologous polynucleotide are alsoconsidered transgenic.

Identification of Plants with an Improved Drought Tolerance Phenotype

We used an Arabidopsis activation tagging screen to identify theassociation between the OBP1 gene (Zhang et al. 1995, supra), which wehave designated “DRO2 (for Drought tolerant),” and an improved droughttolerance phenotype. Briefly, and as further described in the Examples,a large number of Arabidopsis plants were mutated with the pSKI015vector, which comprises a T-DNA from the Ti plasmid of Agrobacteriumtumijaciens, a viral enhancer element, and a selectable marker gene(Weigel et al, supra). When the T-DNA inserts into the genome oftransformed plants, the enhancer element can cause up-regulation genesin the vicinity, generally within about 10 kilobase (kb) of theinsertion. Ti plants were exposed to the selective agent in order tospecifically recover transformed plants that expressed the selectablemarker and therefore harbored T-DNA insertions. Samples of approximately18 seeds were planted, grown for four weeks under adequate waterconditions, and then deprived of water for 10-14 days. Plants that didnot wilt, and that maintained a high water content were identified asdrought tolerant.

An Arabidopsis line that showed increased drought tolerance wasidentified. The association of the DRO2 gene with the drought tolerancephenotype was discovered by analysis of the genomic DNA sequenceflanking the T-DNA insertion in the identified line. Accordingly, DRO2genes and/or polypeptides may be employed in the development ofgenetically modified plants having a modified drought tolerancephenotype (“a DRO2 phenotype”). DRO2 genes may be used in the generationof crops and/or other plant species that have improved ability tosurvive in low-water conditions. The DRO2 phenotype may further enhancethe overall health of the plant.

DRO2 Nucleic Acids and Polypeptides

Arabidopsis DRO2 nucleic acid (cDNA) sequence is provided in SEQ ID NO:1and in Genbank entry GI 7630025, nucleotides 110251-111012. Thecorresponding protein sequence is provided in SEQ ID NO:2 and in GI7630027. A variant polypeptide, encoded by the same genomic sequence, isprovided in GI 1212759 (Zhang et al. 1995, supra).

As used herein, the term “DRO2 polypeptide” refers to a full-length DRO2protein or a fragment, derivative (variant), or ortholog thereof that is“functionally active,” meaning that the protein fragment, derivative, orortholog exhibits one or more or the functional activities associatedwith the polypeptide of SEQ ID NO:2. In one preferred embodiment, afunctionally active DRO2 polypeptide causes an altered drought tolerancephenotype when mis-expressed in a plant. In a further preferredembodiment, misexpression of the functionally active DRO2 polypeptidecauses improved drought tolerance. In another embodiment, a functionallyactive DRO2 polypeptide is capable of rescuing defective (includingdeficient) endogenous DRO2 activity when expressed in a plant or inplant cells; the rescuing polypeptide may be from the same or from adifferent species as that with defective activity. In anotherembodiment, a functionally active fragment of a full length DRO2polypeptide (i.e., a native polypeptide having the sequence of SEQ IDNO:2 or a naturally occurring ortholog thereof) retains one of more ofthe biological properties associated with the full-length DRO2polypeptide, such as signaling activity, binding activity, catalyticactivity, or cellular or extra-cellular localizing activity. PreferredDRO2 polypeptides display DNA-binding activity. A DRO2 fragmentpreferably comprises a DRO2 domain, such as a C- or N-terminal orcatalytic domain, among others, and preferably comprises at least 10,preferably at least 20, more preferably at least 25, and most preferablyat least 50 contiguous amino acids of a DRO2 protein. Functional domainscan be identified using the PFAM program (Bateman A et al., NucleicAcids Res (1999) 27:260-262; website at pfam.wustl.edu). A preferredDRO2 fragment comprises a DNA binding domain, most preferably a Dof-typezinc finger domain (PF02701). Functionally active variants offull-length DRO2 polypeptides or fragments thereof include polypeptideswith amino acid insertions, deletions, or substitutions that retain oneof more of the biological properties associated with the full-lengthDRO2 polypeptide. In some cases, variants are generated that change thepost-translational processing of a DRO2 polypeptide. For instance,variants may have altered protein transport or protein localizationcharacteristics or altered protein half-life compared to the nativepolypeptide.

As used herein, the term “DRO2 nucleic acid” encompasses nucleic acidswith the sequence provided in or complementary to the sequence providedin SEQ ID NO:1, as well as functionally active fragments, derivatives,or orthologs thereof. A DRO2 nucleic acid of this invention may be DNA,derived from genomic DNA or cDNA, or RNA.

In one embodiment, a functionally active DRO2 nucleic acid encodes or iscomplementary to a nucleic acid that encodes a functionally active DRO2polypeptide. Included within this definition is genomic DNA that servesas a template for a primary RNA transcript (i.e., an mRNA precursor)that requires processing, such as splicing, before encoding thefunctionally active DRO2 polypeptide. A DRO2 nucleic acid can includeother non-coding sequences, which may or may not be transcribed; suchsequences include 5′ and 3′ UTRs, polyadenylation signals and regulatorysequences that control gene expression, among others, as are known inthe art. Some polypeptides require processing events, such asproteolytic cleavage, covalent modification, etc., in order to becomefully active. Accordingly, functionally active nucleic acids may encodethe mature or the pre-processed DRO2 polypeptide, or an intermediateform. A DRO2 polynucleotide can also include heterologous codingsequences, for example, sequences that encode a marker included tofacilitate the purification of the fused polypeptide, or atransformation marker.

In another embodiment, a functionally active DRO2 nucleic acid iscapable of being used in the generation of loss-of-function DRO2phenotypes, for instance, via antisense suppression, co-suppression,etc.

In one preferred embodiment, a DRO2 nucleic acid used in the methods ofthis invention comprises a nucleic acid sequence that encodes or iscomplementary to a sequence that encodes a DRO2 polypeptide having atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identityto the polypeptide sequence presented in SEQ ID NO:2.

In another embodiment a DRO2 polypeptide of the invention comprises apolypeptide sequence with at least 50% or 60% identity to the DRO2polypeptide sequence of SEQ ID NO:2, and may have at least 70%, 80%,85%, 90% or 95% or more sequence identity to the DRO2 polypeptidesequence of SEQ ID NO:2. In another embodiment, a DRO2 polypeptidecomprises a polypeptide sequence with at least 50%, 60%, 70%, 80%, 85%,90% or 95% or more sequence identity to a functionally active fragmentof the polypeptide presented in SEQ ID NO:2, such as a DNA bindingdomain. In yet another embodiment, a DRO2 polypeptide comprises apolypeptide sequence with at least 50%, 60%, 70%, 80%, or 90% identityto the polypeptide sequence of SEQ ID NO:2 over its entire length andcomprises a DNA binding domain, most preferably a Dof-type zinc fingerdomain.

In another aspect, a DRO2 polynucleotide sequence is at least 50% to 60%identical over its entire length to the DRO2 nucleic acid sequencepresented as SEQ ID NO:1, or nucleic acid sequences that arecomplementary to such a DRO2 sequence, and may comprise at least 70%,80%, 85%, 90% or 95% or more sequence identity to the DRO2 sequencepresented as SEQ ID NO:1 or a functionally active fragment thereof, orcomplementary sequences.

As used herein, “percent (%) sequence identity” with respect to aspecified subject sequence, or a specified portion thereof, is definedas the percentage of nucleotides or amino acids in the candidatederivative sequence identical with the nucleotides or amino acids in thesubject sequence (or specified portion thereof), after aligning thesequences and introducing gaps, if necessary to achieve the maximumpercent sequence identity, as generated by the program WU-BLAST-2.0a19(Altschul et al., J. Mol. Biol., (1990) 215:403-410; website atblast.wustl.edu/blast/README.html) with search parameters set to defaultvalues. The HSP S and HSP S2 parameters are dynamic values and areestablished by the program itself depending upon the composition of theparticular sequence and composition of the particular database againstwhich the sequence of interest is being searched. A “% identity value”is determined by the number of matching identical nucleotides or aminoacids divided by the sequence length for which the percent identity isbeing reported. “Percent (%) amino acid sequence similarity” isdetermined by doing the same calculation as for determining % amino acidsequence identity, but including conservative amino acid substitutionsin addition to identical amino acids in the computation. A conservativeamino acid substitution is one in which an amino acid is substituted foranother amino acid having similar properties such that the folding oractivity of the protein is not significantly affected. Aromatic aminoacids that can be substituted for each other are phenylalanine,tryptophan, and tyrosine; interchangeable hydrophobic amino acids areleucine, isoleucine, methionine, and valine; interchangeable polar aminoacids are glutamine and asparagine; interchangeable basic amino acidsare arginine, lysine and histidine; interchangeable acidic amino acidsare aspartic acid and glutamic acid; and interchangeable small aminoacids are alanine, serine, threonine, cysteine and glycine.

Derivative nucleic acid molecules of the subject nucleic acid moleculesinclude sequences that hybridize to the nucleic acid sequence of SEQ IDNO:1. The stringency of hybridization can be controlled by temperature,ionic strength, pH, and the presence of denaturing agents such asformamide during hybridization and washing. Conditions routinely usedare well known (see, e.g., Current Protocol in Molecular Biology, Vol.1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al.,supra). In some embodiments, a nucleic acid molecule of the invention iscapable of hybridizing to a nucleic acid molecule containing thenucleotide sequence of SEQ ID NO:1 under stringent hybridizationconditions that comprise: prehybridization of filters containing nucleicacid for 8 hours to overnight at 65° C. in a solution comprising6×single strength citrate (SSC) (1×SSC is 0.15 M NaCl, 0.015 M Nacitrate; pH 7.0), 5×Denhardt's solution, 0.05% sodium pyrophosphate and100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C. ina solution containing 6×SSC, 1×Denhardt's solution, 100 μg/ml yeast tRNAand 0.05% sodium pyrophosphate; and washing of filters at 65° C. for 1 hin a solution containing 0.2×SSC and 0.1% SDS (sodium dodecyl sulfate).In other embodiments, moderately stringent hybridization conditions areused that comprise: pretreatment of filters containing nucleic acid for6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mMTris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C. ina solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA,and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hourat 55° C. in a solution containing 2×SSC and 0.1% SDS. Alternatively,low stringency conditions can be used that comprise: incubation for 8hours to overnight at 37° C. in a solution comprising 20% formamide,5×SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured sheared salmon sperm DNA;hybridization in the same buffer for 18 to 20 hours; and washing offilters in 1×SSC at about 37° C. for 1 hour.

As a result of the degeneracy of the genetic code, a number ofpolynucleotide sequences encoding a DRO2 polypeptide can be produced.For example, codons may be selected to increase the rate at whichexpression of the polypeptide occurs in a particular host species, inaccordance with the optimum codon usage dictated by the particular hostorganism (see, e.g., Nakamura Y et al, Nucleic Acids Res (1999) 27:292).Such sequence variants may be used in the methods of this invention.

The methods of the invention may use orthologs of the Arabidopsis DRO2.Methods of identifying the orthologs in other plant species are known inthe art. Normally, orthologs in different species retain the samefunction, due to presence of one or more protein motifs and/or3-dimensional structures. In evolution, when a gene duplication eventfollows speciation, a single gene in one species, such as Arabidopsis,may correspond to multiple genes (paralogs) in another. As used herein,the term “orthologs” encompasses paralogs. When sequence data isavailable for a particular plant species, orthologs are generallyidentified by sequence homology analysis, such as BLAST analysis,usually using protein bait sequences. Sequences are assigned as apotential ortholog if the best hit sequence from the forward BLASTresult retrieves the original query sequence in the reverse BLAST(Huynen M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et al., Genome Research (2000) 10:1204-1210). Programs for multiplesequence alignment, such as CLUSTAL (Thompson J D et al., Nucleic AcidsRes (1994) 22:4673-4680) may be used to highlight conserved regionsand/or residues of orthologous proteins and to generate phylogenetictrees. In a phylogenetic tree representing multiple homologous sequencesfrom diverse species (e.g., retrieved through BLAST analysis),orthologous sequences from two species generally appear closest on thetree with respect to all other sequences from these two species.Structural threading or other analysis of protein folding (e.g., usingsoftware by ProCeryon, Biosciences, Salzburg, Austria) may also identifypotential orthologs. Nucleic acid hybridization methods may also be usedto find orthologous genes and are preferred when sequence data are notavailable. Degenerate PCR and screening of cDNA or genomic DNA librariesare common methods for finding related gene sequences and are well knownin the art (see, e.g., Sambrook, supra; Dieffenbach C and Dveksler G(Eds.) PCR Primer: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, NY, 1989). For instance, methods for generating a cDNA libraryfrom the plant species of interest and probing the library withpartially homologous gene probes are described in Sambrook et al. Ahighly conserved portion of the Arabidopsis DRO2 coding sequence may beused as a probe. DRO2 ortholog nucleic acids may hybridize to thenucleic acid of SEQ ID NO:1 under high, moderate, or low stringencyconditions. After amplification or isolation of a segment of a putativeortholog, that segment may be cloned and sequenced by standardtechniques and utilized as a probe to isolate a complete cDNA or genomicclone. Alternatively, it is possible to initiate an EST project togenerate a database of sequence information for the plant species ofinterest. In another approach, antibodies that specifically bind knownDRO2 polypeptides are used for ortholog isolation. Western blot analysiscan determine that a DRO2 ortholog (i.e., an orthologous protein) ispresent in a crude extract of a particular plant species. Whenreactivity is observed, the sequence encoding the candidate ortholog maybe isolated by screening expression libraries representing theparticular plant species. Expression libraries can be constructed in avariety of commercially available vectors, including lambda gt11, asdescribed in Sambrook, et al., supra. Once the candidate ortholog(s) areidentified by any of these means, candidate orthologous sequence areused as bait (the “query”) for the reverse BLAST against sequences fromArabidopsis or other species in which DRO2 nucleic acid and/orpolypeptide sequences have been identified.

DRO2 nucleic acids and polypeptides may be obtained using any availablemethod. For instance, techniques for isolating cDNA or genomic DNAsequences of interest by screening DNA libraries or by using polymerasechain reaction (PCR), as previously described, are well known in theart. Alternatively, nucleic acid sequence may be synthesized. Any knownmethod, such as site directed mutagenesis (Kunkel T A et al., MethodsEnzymol. (1991) 204:125-39), may be used to introduce desired changesinto a cloned nucleic acid.

In general, the methods of the invention involve incorporating thedesired form of the DRO2 nucleic acid into a plant expression vector fortransformation of in plant cells, and the DRO2 polypeptide is expressedin the host plant.

An isolated DRO2 nucleic acid molecule is other than in the form orsetting in which it is found in nature and is identified and separatedfrom least one contaminant nucleic acid molecule with which it isordinarily associated in the natural source of the DRO2 nucleic acid.However, an isolated DRO2 nucleic acid molecule includes DRO2 nucleicacid molecules contained in cells that ordinarily express DRO2 where,for example, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

Generation of Genetically Modified Plants with a Drought TolerancePhenotype

DRO2 nucleic acids and polypeptides may be used in the generation ofgenetically modified plants having a modified, preferably an improveddrought tolerance phenotype. Such plants may further display increasedtolerance to other abiotic stresses, particular salt-stress andfreezing, as responses to these stresses and drought stress are mediatedby ABA (Thomashow, 1999 Annu. Revl Plant Physiol. Plant Mol. Biol. 50:571; Cushman and Bohnert, 2000, Curr. Opin. Plant Biol. 3: 117; Kang etal. 2002, Plant Cell 14:343-357; Quesada et al. 2000, Genetics 154: 421;Kasuga et al. 1999, Nature Biotech. 17: 287-291).

The methods described herein are generally applicable to all plants.Drought tolerance is an important trait in almost any agricultural crop;most major agricultural crops, including corn, wheat, soybeans, cotton,alfalfa, sugar beets, onions, tomatoes, and beans, are susceptible todrought stress. Although activation tagging and gene identification arecarried out in Arabidopsis, the DRO2 gene (or an ortholog, variant orfragment thereof) may be expressed in any type of plant. The inventionmay directed to fruit- and vegetable-bearing plants, plants used in thecut flower industry, grain-producing plants, oil-producing plants,nut-producing plants, crops including corn (Zea mays), soybean (Glycinemax), cotton (Gossypium), tomato (Lycopersicum esculentum), alfalfa(Medicago sativa), flax (Linum usitatissimumy, tobacco (Nicotiana), andturfgrass (Poaceae family), and other forage crops, among others.

The skilled artisan will recognize that a wide variety of transformationtechniques exist in the art, and new techniques are continually becomingavailable. Any technique that is suitable for the target host plant canbe employed within the scope of the present invention. For example, theconstructs can be introduced in a variety of forms including, but notlimited to as a strand of DNA, in a plasmid, or in an artificialchromosome. The introduction of the constructs into the target plantcells can be accomplished by a variety of techniques, including, but notlimited to Agrobacterium-mediated transformation, electroporation,microinjection, microprojectile bombardment calcium-phosphate-DNAco-precipitation or liposome-mediated transformation of a heterologousnucleic acid. The transformation of the plant is preferably permanent,i.e. by integration of the introduced expression constructs into thehost plant genome, so that the introduced constructs are passed ontosuccessive plant generations. Depending upon the intended use, aheterologous nucleic acid construct comprising a DRO2 polynucleotide mayencode the entire protein or a biologically active portion thereof.

In one embodiment, binary Ti-based vector systems may be used totransfer polynucleotides. Standard Agrobacterium binary vectors areknown to those of skill in the art, and many are commercially available(e.g., pBI121 Clontech Laboratories, Palo Alto, Calif.).

The optimal procedure for transformation of plants with Agrobacteriumvectors will vary with the type of plant being transformed. Exemplarymethods for Agrobacterium-mediated transformation include transformationof explants of hypocotyl, shoot tip, stem or leaf tissue, derived fromsterile seedlings and/or plantlets. Such transformed plants may bereproduced sexually, or by cell or tissue culture. Agrobacteriumtransformation has been previously described for a large number ofdifferent types of plants and methods for such transformation may befound in the scientific literature.

Expression (including transcription and translation) of DRO2 may beregulated with respect to the level of expression, the tissue type(s)where expression takes place and/or developmental stage of expression. Anumber of heterologous regulatory sequences (e.g., promoters andenhancers) are available for controlling the expression of a DRO2nucleic acid. These include constitutive, inducible and regulatablepromoters, as well as promoters and enhancers that control expression ina tissue- or temporal-specific manner. Exemplary constitutive promotersinclude the raspberry E4 promoter (U.S. Pat. Nos. 5,783,393 and5,783,394), the 35S CaMV (Jones J D et al, (1992) Transgenic Res1:285-297), the CsVMV promoter (Verdaguer B et al., Plant Mol Biol(1998) 37:1055-1067) and the melon actin promoter (published PCTapplication WO0056863). Exemplary tissue-specific promoters include thetomato E4 and E8 promoters (U.S. Pat. No. 5,859,330) and the tomato 2AIIgene promoter (Van Haaren M J J et al., Plant Mol Bio (1993)21:625-640).

In one preferred embodiment, DRO2 expression is under control ofregulatory sequences from genes whose expression is associated withdrought stress. For example, when the promoter of the drought stressresponsive Arabidopsis rd29A gene was used to drive expression ofDREB1A, Arabidopsis plants were more tolerant to drought, salt andfreezing stress and did not have the stunted stature associated withplants over-expressing the DREB1A gene from the CaMV 35S promoter(Kasuga et al, 1999 Nature Biotech 17:287). Promoters from otherArabidopsis genes that are responsive to drought stress, such as COR47(Welin et al. 1995, Plant Mol. Biol. 29: 391), KIN1 (Kurkela and Franck,1990, Plant Mol. Biol. 15: 137), RD22BP (Abe et al. 1997, Plant Cell 9,1859), ABA1 (Accession Number AAG17703), and ABA3 (Xiong et al. 2001,Plant Cell 13: 2063), could be used. Promoters from drought stressinducible genes in other species could be used also. Examples are therab17, ZmFer1 and ZmFer2 genes from maize (Bush et al, 1997 Plant J11:1285; Fobis-Loisy, 1995 Eur J Biochem 231:609), the tdi-65 gene fromtomato (Harrak, 2001 Genome 44:368), the His1 gene of tobacco (Wei andO'Connell, 1996 Plant Mol Biol 30:255), the Vupat1 gene from cowpea(Matos, 2001 FEBS Lett 491:188), and CDSP34 from Solanum tuberosum(Gillet et al, 1998 Plant J 16:257).

In yet another aspect, in some cases it may be desirable to inhibit theexpression of endogenous DRO2 in a host cell. Exemplary methods forpracticing this aspect of the invention include, but are not limited toantisense suppression (Smith, et al., Nature (1988) 334:724-726; van derKrol et al., Biotechniques (1988) 6:958-976); co-suppression (Napoli, etal, Plant Cell (1990) 2:279-289); ribozymes (PCT Publication WO97/10328); and combinations of sense and antisense (Waterhouse, et al.,Proc. Natl. Acad. Sci. USA (1998) 95:13959-13964). Methods for thesuppression of endogenous sequences in a host cell typically employ thetranscription or transcription and translation of at least a portion ofthe sequence to be suppressed. Such sequences may be homologous tocoding as well as non-coding regions of the endogenous sequence.Antisense inhibition may use the entire cDNA sequence (Sheehy et al.,Proc. Natl. Acad. Sci. USA (1988) 85:8805-8809), a partial cDNA sequenceincluding fragments of 5′ coding sequence, (Cannon et al., Plant Molec.Biol. (1990) 15:39-47), or 3′ non-coding sequences (Ch'ng et al., Proc.Natl. Acad. Sci. USA (1989) 86:10006-10010). Cosuppression techniquesmay use the entire cDNA sequence (Napoli et al., supra; van der Krol etal., The Plant Cell (1990) 2:291-299), or a partial cDNA sequence (Smithet al., Mol. Gen. Genetics (1990) 224:477-481).

Standard molecular and genetic tests may be performed to further analyzethe association between a gene and an observed phenotype. Exemplarytechniques are described below.

1. DNA/RNA Analysis

The stage- and tissue-specific gene expression patterns in mutant versuswild-type lines may be determined, for instance, by in situhybridization. Analysis of the methylation status of the gene,especially flanking regulatory regions, may be performed. Other suitabletechniques include overexpression, ectopic expression, expression inother plant species and gene knock-out (reverse genetics, targetedknock-out, viral induced gene silencing [VIGS, see Baulcombe D, ArchVirol Suppl (1999) 15:189-201]).

In a preferred application expression profiling, generally by microarray analysis, is used to simultaneously measure differences or inducedchanges in the expression of many different genes. Techniques formicroarray analysis are well known in the art (Schena M et al., Science(1995) 270:467-470; Baldwin D et al., (1999) Cur Opin Plant Biol.2(2):96-103; Dangond F, Physiol Genomics (2000) 2:53-58; van Hal N L etal., J Biotechnol (2000) 78:271-280; Richmond T and Somerville S, CurrOpin Plant Biol (2000) 3:108-116). Expression profiling of individualtagged lines may be performed. Such analysis can identify other genesthat are coordinately regulated as a consequence of the overexpressionof the gene of interest, which may help to place an unknown gene in aparticular pathway.

2. Gene Product Analysis

Analysis of gene products may include recombinant protein expression,antisera production, immunolocalization, biochemical assays forcatalytic or other activity, analysis of phosphorylation status, andanalysis of interaction with other proteins via yeast two-hybrid assays.

3. Pathway Analysis

Pathway analysis may include placing a gene or gene product within aparticular biochemical, metabolic or signaling pathway based on itsmis-expression phenotype or by sequence homology with related genes.Alternatively, analysis may comprise genetic crosses with wild-typelines and other mutant lines (creating double mutants) to order the genein a pathway, or determining the effect of a mutation on expression ofdownstream “reporter” genes in a pathway.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention. All publications cited herein are expressly incorporatedherein by reference for the purpose of describing and disclosingcompositions and methodologies that might be used in connection with theinvention. All cited patents, patent applications, and sequenceinformation in referenced websites and public databases are alsoincorporated by reference.

EXAMPLES Example 1 Generation of Plants with a DRO2 Phenotype byTransformation with an Activation Tagging Construct

Mutants were generated using the activation tagging “ACTTAG” vector,pSKI015 (GI 6537289; Weigel D et al., supra). Standard methods were usedfor the generation of Arabidopsis transgenic plants, and wereessentially as described in published application PCT WO0183697.Briefly, T0 Arabidopsis (Col-0) plants were transformed withAgrobacterium carrying the pSKI015 vector, which comprises T-DNA derivedfrom the Agrobacterium Ti plasmid, an herbicide resistance selectablemarker gene, and the 4×CaMV 35S enhancer element. Transgenic plants wereselected at the T1 generation based on herbicide resistance. T2 seed wascollected from T1 plants and stored in an indexed collection, and aportion of the T2 seed was accessed for the screen.

Approximately 18 T2 seed from each of line tested were planted in soil.The seed were stratified for 3 days at 4° C. and then grown in thegreenhouse for 4 weeks with adequate water. After this time, water waswithheld for 10-14 days until most of the plants were severely wilted.T2 lines containing plants that appeared to be non-wilted wereidentified and their relative water content (RWC) measured. RWC wascalculated from the following equation:RWC=(Fresh Weight-Dry Weight)/(Turgid Weight-Dry Weight)×100.

For RWC analysis, 2-3 leaves were taken from each drought tolerant(non-wilted) individual within a line and pooled. Fresh Weight is themass of these leaves immediately after the drought treatment. Turgidweight is the mass of these leaves after being rehydrated for 24 h inwater. Dry Weight is the mass of these leaves after being air-dried.Control plants had RWC values of approximately 26% while. Plants thathad RWC values of at least 50% were identified as drought tolerant.

After identification in the primary screen, putative mutant (i.e.,drought-tolerant) lines were re-screened using the protocol describedabove, with the exception that 18 seeds were planted individually.

The ACTTAG line designated W000017146 was identified as having adominant drought tolerance phenotype. In the secondary screen, 14 of the18 plants tested had RWC values of at least 50%.

Example 2 Characterization of the T-DNA Insertion in Plants Exhibitingthe Altered Drought Tolerance Phenotype

We performed standard molecular analyses, essentially as described inpatent application PCT WO0183697, to determine the site of the T-DNAinsertion associated with the increased drought tolerance phenotype.Briefly, genomic DNA was extracted from plants exhibiting increaseddrought tolerance. PCR, using primers specific to the pSKI015 vector,confirmed the presence of the 35S enhancer in plants from lineW000017146, and Southern blot analysis verified the genomic integrationof the ACTTAG T-DNA and showed the presence of a single T-DNA insertionin the transgenic line.

Inverse PCR was used to recover genomic DNA flanking the T-DNAinsertion, which was then subjected to sequence analysis.

The sequence flanking the left T-DNA border was subjected to a basicBLASTN search and/or a search of the Arabidopsis Information Resource(TAIR) database (available at the Arabidopsis.org website), whichrevealed sequence identity to F11C1 (GI 7630025), mapped to chromosome3. Sequence analysis revealed that the T-DNA had inserted in thevicinity (i.e., within about 10 kb) of the gene whose nucleotidesequence is presented as SEQ ID NO:1 and GI 7630025, nucleotides110251-111012, and which we designated DRO2. This insertion appearedcomplex; while the upstream end had a normal left border, the downstreamend was associated with a fragment of the left border, in addition tothe right border, and a small genomic deletion. The right side of theT-DNA insertion (and the right border) was approximately 1.5 kb 5′ tothe start codon of SEQ ID NO:1.

Example 3 Analysis of Arabidopsis DRO2 Sequence

The amino acid sequence predicted from the DRO2 nucleic acid sequence ispresented in SEQ ID NO:2 and GI 7630027.

Sequence analyses were performed with BLAST (Altschul et al., 1990, J.Mol. Biol. 215:403-410) and PFAM (Bateman et al., supra), PSORT (NakaiK, and Horton P, 1999, Trends Biochem Sci 24:34-6), among others. PFAManalysis predicted a “Dof (DNA binding with one finger)” zinc fingerDNA-binding domain (PF02701; Shimofurutani et al. 1998, FEBS Lett430:251-256). Dof proteins are a class of transcription factors uniqueto plants (Yanagisawa, 1996, Biochem Mol Biol Int 38:665-73), containinga highly conserved 52 amino acid motif containing a single Zinc finger(CX₂CX₂₁CX₂C). The Dof domain of SEQ ID NO:2 is at amino acids 25-87.

Example 4 Confirmation of Phenotype/Genotype Association

RT-PCR analysis showed that the DRO2 gene was specifically overexpressedin plants from the line displaying the improved drought tolerancephenotype. Specifically, RNA was extracted from tissues derived fromplants exhibiting the DRO2 phenotype and from wild type COL-0 plants.RT-PCR was performed using primers specific to the sequence presented asSEQ ID NO:1 and to other predicted genes in the vicinity of the TDNAinsertion. The results showed that plants displaying the DRO2 phenotypeoverexpressed the mRNA for the DRO2 gene, indicating the enhancedexpression of the DRO2 gene is correlated with the DRO2 phenotype.

1. A method, comprising: identifying a plant with defective endogenousDRO2 activity; transforming the plant having defective endogenous DRO2activity with a plant transformation vector comprising a heterologousconstitutive promoter operatively linked to a nucleotide sequence thatencodes a DRO2 polypeptide comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:2,wherein the heterologous constitutive promoter provides overexpressionof a DRO2 transcript encoding said DRO2 polypeptide; subjecting thetransformed plant to water deprivation; and measuring the relative watercontent of the transformed plant, wherein an increase in relative watercontent of the transformed plant as compared to a control plant whichdid not receive the plant transformation vector indicates that thetransformed plant has increased drought tolerance, and thereby impartingdrought tolerance to the plant which had defective endogenous DRO2activity prior to said transformation.
 2. The method of claim 1, whereinsubjecting the transformed plant to water deprivation comprisesdepriving the transformed plant of water for at least 10 days.
 3. Themethod of claim 1, wherein the polypeptide consists of an amino acidsequence having at least 95% sequence identity with SEQ ID NO:2.
 4. Themethod of claim 1, wherein the polypeptide comprises the amino acidsequence set forth in SEQ ID NO:2.
 5. The method of claim 1, wherein thepolypeptide consists of the amino acid sequence set forth in SEQ IDNO:2.